In vitro methods for evaluating the in vivo effectiveness of dosage forms of microparticulate of nanoparticulate active agent compositions

ABSTRACT

Disclosed are in vitro methods for evaluating the in vivo redispersibility of dosage forms of poorly water-soluble active agents. The methods utilize media representative of in vivo human physiological conditions.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.10/075,443, filed on Feb. 15, 2002 now U.S. Pat. No. 6,592,903, which isa continuation of application Ser. No. 09/666,539, filed on Sep. 21,2000, now U.S. Pat. No. 6,375,986.

FIELD OF THE INVENTION

The present invention is directed to in vitro methods of evaluating thein vivo effectiveness of dosage forms of microparticulate ornanoparticulate poorly water-soluble active agent compositions. Themethods comprise evaluating the redispersibility of dosage forms ofmicroparticulate or nanoparticulate active agents in a biorelevantaqueous medium that preferably mimics in vivo human physiologicalconditions.

BACKGROUND OF THE INVENTION

A. Background Regarding Conventional in vitro Methods for Evaluating thein vivo Effectiveness of Dosage Forms of Active Agents

Active agents are marketed in a wide variety of dosage forms, solid,semi-solid, or liquid dosage formulations, immediate release dosageforms, modified release dosage forms, extended release dosage forms,delayed release dosage forms, pulsatile release dosage forms, controlledrelease dosage forms, fast melt (“waterless”) tablet formulations, drypowders for oral suspension or pulmonary administration,multiparticulates, sprinkles, tablets, capsules and related solidpresentations for oral administration, lyophilized formulations,sachets, lozenges, syrups, liquids for injection or oral delivery, etc.

For an active agent to exhibit pharmacological activity, the activeagent must dissolve and be absorbed by the patient. If the active agentdoes not dissolve, absorption will not occur and pharmacologicalactivity will not be achieved. Upon administration of a dosage form,several events must occur prior to dissolution and subsequent absorptionof the active agent: (1) the dosage form must disintegrate, (2) disperseinto small particles; (3) dissolve in its molecular form (4) followed byabsorption. If an active agent is not dispersed sufficiently it will notbe dissolved readily and consequently will pass through thegastrointestinal tract of the patient, resulting in low bioavailabilityof the administered active agent.

Conventional in vitro analytical methodologies for evaluating the invivo effectiveness of poorly water-soluble active agents attempt toassess product quality by measuring the rate and extent of active agentdissolution in an aqueous medium, and generally in the presence ofsurfactants or cosolvents. See e.g., Umesh V. Banakar, PharmaceuticalDissolution Testing, Drugs and Pharmaceutical Sciences, Vol. 49 (1992).Such aggressive solubilizing agents can decrease the sensitivity of theanalytical test. Moreover, such dissolution tests are conducted in mediawhich are generally not reflective of in vivo human physiologicalconditions and do not measure the dosage form's redispersibilityqualities. See e.g., J. T. Carstensen, Pharmaceutical Principles ofSolid Dosage Forms, pp. 10–11 (Technomic Publishing Co., Inc. (1993);Schmidt et al., “Incorporation of Polymeric Nanoparticles into SolidDosage Forms,” J. Control Release, 57 (2): 115–25 (1999). See alsoVolker Bühler, Generic Drug Formulations, Section 4.3 (Fine Chemicals,2^(nd) Edition, 1998). See De Jaeghere et al., “pH-Dependent DissolvingNano- and Microparticles for Improved Peroral Delivery of a HighlyLipophilic Compound in Dogs,” AAPS PharmSci., 3:8 (February 2001).

Following disintegration, the next step in making an active agentbioavailable from a nanoparticulate or microparticulate dosage form isthe redispersibility of the formulation back to the originalnanoparticulate or microparticulate active agent particle size presentbefore the active agent was formulated into a dosage form. Therefore,the ability of a method to quantitate that part of the process is atleast as important as the actual dissolution of the active agent. Infact, this part of the process is more relevant for poorly solublecomponents than the last step of the process in predicting in vivobioavailability.

B. Background Regarding Nanoparticulate Compositions

Nanoparticulate compositions, first described in U.S. Pat. No. 5,145,684(“the '684 patent”), are particles consisting of a poorly soluble activeagent having adsorbed onto the surface thereof a non-crosslinked surfacestabilizer. The '684 patent also describes methods of making suchnanoparticulate compositions. Nanoparticulate compositions are desirablebecause with a decrease in particle size, and a consequent increase insurface area, a composition can exhibit superior bioavailability.

Redispersibility properties of a nanoparticulate based dosage form areespecially important, since when the dosage form of a nanoparticulateactive agent does not suitably redisperse following administration, thebenefits of formulating the active agent into nanoparticles may becompromised or altogether lost. This is because in the absence ofredispersibility the dosage form produces clumps or large aggregates ofparticles, and not discrete nanoparticles of active agent.

Methods of making nanoparticulate compositions are described, forexample, in U.S. Pat. Nos. 5,518,187 and 5,862,999, both for “Method ofGrinding Pharmaceutical Substances;” U.S. Pat. No. 5,718,388, for“Continuous Method of Grinding Pharmaceutical Substances;” and U.S. Pat.No. 5,510,118 for “Process of Preparing Therapeutic CompositionsContaining Nanoparticles.”

Nanoparticulate compositions are also described, for example, in U.S.Pat. No. 5,298,262 for “Use of Ionic Cloud Point Modifiers to PreventParticle Aggregation During Sterilization;” U.S. Pat. No. 5,302,401 for“Method to Reduce Particle Size Growth During Lyophilization;” U.S. Pat.No. 5,318,767 for “X-Ray Contrast Compositions Useful in MedicalImaging;” U.S. Pat. No. 5,326,552 for “Novel Formulation ForNanoparticulate X-Ray Blood Pool Contrast Agents Using High MolecularWeight Non-ionic Surfactants;” U.S. Pat. No. 5,328,404 for “Method ofX-Ray Imaging Using Iodinated Aromatic Propanedioates;” U.S. Pat. No.5,336,507 for “Use of Charged Phospholipids to Reduce NanoparticleAggregation;” U.S. Pat. No. 5,340,564 for “Formulations Comprising Olin10-G to Prevent Particle Aggregation and Increase Stability;” U.S. Pat.No. 5,346,702 for “Use of Non-Ionic Cloud Point Modifiers to MinimizeNanoparticulate Aggregation During Sterilization;” U.S. Pat. No.5,349,957 for “Preparation and Magnetic Properties of Very SmallMagnetic-Dextran Particles;” U.S. Pat. No. 5,352,459 for “Use ofPurified Surface Modifiers to Prevent Particle Aggregation DuringSterilization;” U.S. Pat. Nos. 5,399,363 and 5,494,683, both for“Surface Modified Anticancer Nanoparticles;” U.S. Pat. No. 5,401,492 for“Water Insoluble Non-Magnetic Manganese Particles as Magnetic ResonanceEnhancement Agents;” U.S. Pat. No. 5,429,824 for “Use of Tyloxapol as aNanoparticulate Stabilizer;” U.S. Pat. No. 5,447,710 for “Method forMaking Nanoparticulate X-Ray Blood Pool Contrast Agents Using HighMolecular Weight Non-ionic Surfactants;” U.S. Pat. No. 5,451,393 for“X-Ray Contrast Compositions Useful in Medical Imaging;” U.S. Pat. No.5,466,440 for “Formulations of Oral Gastrointestinal Diagnostic X-RayContrast Agents in Combination with Pharmaceutically Acceptable Clays;”U.S. Pat. No. 5,470,583 for “Method of Preparing NanoparticleCompositions Containing Charged Phospholipids to Reduce Aggregation;”U.S. Pat. No. 5,472,683 for “Nanoparticulate Diagnostic Mixed CarbamicAnhydrides as X-Ray Contrast Agents for Blood Pool and Lymphatic SystemImaging;” U.S. Pat. No. 5,500,204 for “Nanoparticulate Diagnostic Dimersas X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging;”U.S. Pat. No. 5,518,738 for “Nanoparticulate NSAID Formulations;” U.S.Pat. No. 5,521,218 for “Nanoparticulate Iododipamide Derivatives for Useas X-Ray Contrast Agents;” U.S. Pat. No. 5,525,328 for “NanoparticulateDiagnostic Diatrizoxy Ester X-Ray Contrast Agents for Blood Pool andLymphatic System Imaging;” U.S. Pat. No. 5,543,133 for “Process ofPreparing X-Ray Contrast Compositions Containing Nanoparticles;” U.S.Pat. No. 5,552,160 for “Surface Modified NSAID Nanoparticles;” U.S. Pat.No. 5,560,931 for “Formulations of Compounds as NanoparticulateDispersions in Digestible Oils or Fatty Acids;” U.S. Pat. No. 5,565,188for “Polyalkylene Block Copolymers as Surface Modifiers forNanoparticles;” U.S. Pat. No. 5,569,448 for “Sulfated Non-ionic BlockCopolymer Surfactant as Stabilizer Coatings for NanoparticleCompositions;” U.S. Pat. No. 5,571,536 for “Formulations of Compounds asNanoparticulate Dispersions in Digestible Oils or Fatty Acids;” U.S.Pat. No. 5,573,749 for “Nanoparticulate Diagnostic Mixed CarboxylicAnydrides as X-Ray Contrast Agents for Blood Pool and Lymphatic SystemImaging;” U.S. Pat. No. 5,573,750 for “Diagnostic Imaging X-Ray ContrastAgents;” U.S. Pat. No. 5,573,783 for “Redispersible Nanoparticulate FilmMatrices With Protective Overcoats;” U.S. Pat. No. 5,580,579 for“Site-specific Adhesion Within the GI Tract Using NanoparticlesStabilized by High Molecular Weight, Linear Poly(ethylene Oxide)Polymers;” U.S. Pat. No. 5,585,108 for “Formulations of OralGastrointestinal Therapeutic Agents in Combination with PharmaceuticallyAcceptable Clays;” U.S. Pat. No. 5,587,143 for “Butylene Oxide-EthyleneOxide Block Copolymers Surfactants as Stabilizer Coatings forNanoparticulate Compositions;” U.S. Pat. No. 5,591,456 for “MilledNaproxen with Hydroxypropyl Cellulose as Dispersion Stabilizer;” U.S.Pat. No. 5,593,657 for “Novel Barium Salt Formulations Stabilized byNon-ionic and Anionic Stabilizers;” U.S. Pat. No. 5,622,938 for “SugarBased Surfactant for Nanocrystals;” U.S. Pat. No. 5,628,981 for“Improved Formulations of Oral Gastrointestinal Diagnostic X-RayContrast Agents and Oral Gastrointestinal Therapeutic Agents;” U.S. Pat.No. 5,643,552 for “Nanoparticulate Diagnostic Mixed Carbonic Anhydridesas X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging;”U.S. Pat. No. 5,718,388 for “Continuous Method of GrindingPharmaceutical Substances;” U.S. Pat. No. 5,718,919 for “NanoparticlesContaining the R(-)Enantiomer of Ibuprofen;” U.S. Pat. No. 5,747,001 for“Aerosols Containing Beclomethasone Nanoparticle Dispersions;” U.S. Pat.No. 5,834,025 for “Reduction of Intravenously AdministeredNanoparticulate Formulation Induced Adverse Physiological Reactions;”U.S. Pat. No. 6,045,829 “Nanocrystalline Formulations of HumanImmunodeficiency Virus (HIV) Protease Inhibitors Using CellulosicSurface Stabilizers;” U.S. Pat. No. 6,068,858 for “Methods of MakingNanocrystalline Formulations of Human Immunodeficiency Virus (HIV)Protease Inhibitors Using Cellulosic Surface Stabilizers;” U.S. Pat. No.6,153,225 for “Injectable Formulations of Nanoparticulate Naproxen;”U.S. Pat. No. 6,165,506 for “New Solid Dose Form of NanoparticulateNaproxen;” U.S. Pat. No. 6,221,400 for “Methods of Treating MammalsUsing Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV)Protease Inhibitors;” U.S. Pat. No. 6,264,922 for “Nebulized AerosolsContaining Nanoparticle Dispersions;” U.S. Pat. No. 6,267,989 for“Methods for Preventing Crystal Growth and Particle Aggregation inNanoparticle Compositions;” U.S. Pat. No. 6,270,806 for “Use ofPEG-Derivatized Lipids as Surface Stabilizers for NanoparticulateCompositions;” U.S. Pat. No. 6,316,029 for “Rapidly Disintegrating SolidOral Dosage Form,” U.S. Pat. No. 6,375,986 for “Solid DoseNanoparticulate Compositions Comprising a Synergistic Combination of aPolymeric Surface Stabilizer and Dioctyl Sodium Sulfosuccinate;” U.S.Pat. No. 6,428,814 for “Bioadhesive nanoparticulate compositions havingcationic surface stabilizers;” and U.S. Pat. No. 6,432,381 for “Methodsfor targeting drug delivery to the upper and/or lower gastrointestinaltract,” all of which are specifically incorporated by reference. Inaddition, U.S. Patent Application No. 20020012675 A1, published on Jan.31, 2002, for “Controlled Release Nanoparticulate Compositions,”describes nanoparticulate compositions, and is specifically incorporatedby reference.

Amorphous small particle compositions are described, for example, inU.S. Pat. No. 4,783,484 for “Particulate Composition and Use Thereof asAntimicrobial Agent;” U.S. Pat. No. 4,826,689 for “Method for MakingUniformly Sized Particles from Water-Insoluble Organic Compounds;” U.S.Pat. No. 4,997,454 for “Method for Making Uniformly-Sized Particles FromInsoluble Compounds;” U.S. Pat. No. 5,741,522 for “Ultrasmall,Non-aggregated Porous Particles of Uniform Size for Entrapping GasBubbles Within and Methods;” and U.S. Pat. No. 5,776,496, for“Ultrasmall Porous Particles for Enhancing Ultrasound Back Scatter.”

There is a need in the art for effective in vitro methods of evaluatingthe in vivo effectiveness of dosage forms of microparticulate andnanoparticulate poorly water-soluble active agents. The presentinvention satisfies this need.

SUMMARY OF THE INVENTION

The present invention is directed to the surprising discovery that thein vivo effectiveness of dosage forms of nanoparticulate andmicroparticulate poorly water-soluble active agents can be reliablypredicted by utilizing an in vitro redispersibility test. Theredispersibility test employs biorelevant aqueous media that mimic humanphysiological conditions, rather than aggressive, surfactant-enriched orcosolvent-enriched media that facilitate rapid and complete dissolutionof the active pharmaceutical agent.

The redispersibility test of the invention is a quantitative measure ofthe ability of a formulation to regenerate particle sizes that areoptimum in vivo. Such regenerated particle sizes are generally similarto the primary active agent particle size present prior to formulatingthe active agent into a dosage form. For example, if a nanoparticulateactive agent dispersion is used to make the dosage form, then theprimary active agent particle size is that present in the dispersion.The test employs biorelevant aqueous media which mimic in vivo humanphysiological conditions, such as the ionic strength and pH found invivo. Such biorelevant aqueous media can be electrolyte solutions, suchas HCl or NaCl solutions, or solutions of other salts and acids, orcombinations thereof, which have the desired biorelevantcharacteristics.

The method is a dramatic improvement over prior art methods, as theconventional in vitro method of measuring dissolution of a dosage formin a surfactant-enriched or cosolvent-enriched medium may have nocorrelation with redispersibility observed under in vivo humanphysiological conditions.

Also provided by the invention are dosage forms selected by the methodof the invention, and methods of using such dosage forms.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory and are intended to providefurther explanation of the invention as claimed. Other objects,advantages, and novel features will be readily apparent to those skilledin the art from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the mean pharmacokinetic profiles in fasted humanvolunteers upon oral administration of a single 200-mg dose of twonanoparticulate active agent tablet formulations and a commercialcapsule formulation (reference product); and

FIG. 2: Shows the percent redispersibility in electrolyte solutions, asa function of electrolyte concentration, for a spray driednanoparticulate MAP kinase inhibitor composition.

DETAILED DESCRIPTION OF THE INVENTION

One frequent problem associated with prior art poorly water-solubleactive agent compositions was that upon administration to a patient,such as a human or animal, the active agent composition would notredisperse in vivo to a particle size having optimum characteristics forin vivo performance. As a lack of redispersibility upon administrationmay result in an ineffective dosage form, the ability to predict in vivoredispersibility is critical to successful dosage form design.

Poor redispersibility is particularly problematic for nanoparticulateactive agent compositions, for when the nanoparticulate active agentdosage form fails to redisperse upon administration, the dosage form maylose the benefits afforded by formulating the active agent into ananoparticulate particle size. This is because nanoparticulate activeagent compositions benefit from the small particle size of the activeagent; if the active agent does not redisperse into the small particlesizes upon administration, then “clumps” or agglomerated active agentparticles are formed, owing to the extremely high surface free energy ofthe nanoparticulate system and the thermodynamic driving force toachieve an overall reduction in free energy. With the formation of suchagglomerated particles, the bioavailability of the dosage form may fallwell below that observed with the liquid dispersion form of thenanoparticulate active agent.

Similar benefits can also be obtained by designing a microparticulatedosage form using the method of the invention, as regardless of theparticle size of the component active agent, faster and more completedissolution of a microparticulate active agent dosage form is desirable.

The invention is applicable to in vitro methods for evaluating a widevariety of dosage forms, such as solid, semi-solid, or liquid dosageformulations, immediate release dosage forms, modified release dosageforms, extended release dosage forms, delayed release dosage forms,pulsatile release dosage forms, controlled release dosage forms, fastmelt (“waterless”) tablet formulations, dry powders, such as those fororal suspension or pulmonary or nasal administration, multiparticulates,sprinkles, tablets, capsules and related solid presentations for oraladministration, lyophilized formulations, sachets, lozenges, syrups,liquids for injection or oral delivery, etc. In sum, any dosage form canbe evaluated by the method of the invention, including but not limitedto dosage forms intended for oral, pulmonary, nasal, parenteral, rectal,local, or buccal delivery.

The methods of the invention are directed to in vitro techniques capableof quantifying the rate and extent of active agent redispersibility uponintroduction of particulate poorly water-soluble active agent dosageforms into biorelevant aqueous media. Such biorelevant aqueous media canbe any aqueous media that exhibit the desired ionic strength and pH,which form the basis for the biorelevance of the media. The desired pHand ionic strength are those that are representative of physiologicalconditions found in the human body. Such biorelevant aqueous media canbe, for example, aqueous electrolyte solutions or aqueous solutions ofany salt, acid, or base, or a combination thereof, which exhibit thedesired pH and ionic strength.

Biorelevant pH is well known in the art. For example, in the stomach,the pH ranges from slightly less than 2 (but typically greater than 1)up to 4 or 5. In the small intestine the pH can range from 4 to 6, andin the colon it can range from 6 to 8. Biorelevant ionic strength isalso well known in the art. Fasted state gastric fluid has an ionicstrength of about 0.1M while fasted state intestinal fluid has an ionicstrength of about 0.14. See e.g., Lindahl et al., “Characterization ofFluids from the Stomach and Proximal Jejunum in Men and Women,” Pharm.Res., 14 (4): 497–502 (1997).

It is believed that the pH and ionic strength of the test solution ismore critical than the specific chemical content. Accordingly,appropriate pH and ionic strength values can be obtained throughnumerous combinations of strong acids, strong bases, salts, single ormultiple conjugate acid-base pairs (i.e., weak acids and correspondingsalts of that acid), monoprotic and polyprotic electrolytes, etc.

Representative electrolyte solutions can be, but are not limited to, HClsolutions, ranging in concentration from about 0.001 to about 0.1 M, andNaCl solutions, ranging in concentration from about 0.001 to about 0.1M, and mixtures thereof. For example, electrolyte solutions can be, butare not limited to, about 0.1 M HCl or less, about 0.01 M HCl or less,about 0.001 M HCl or less, about 0.1 M NaCl or less, about 0.01 M NaClor less, about 0.001 M NaCl or less, and mixtures thereof. Of theseelectrolyte solutions, 0.01 M HCl and/or 0.1 M NaCl, are mostrepresentative of fasted human physiological conditions, owing to the pHand ionic strength conditions of the proximal gastrointestinal tract.

Electrolyte concentrations of 0.001 M HCl, 0.01 M HCl, and 0.1 M HClcorrespond to pH 3, pH 2, and pH 1, respectively. Thus, a 0.01 M HClsolution simulates typical acidic conditions found in the stomach. Asolution of 0.1 M NaCl provides a reasonable approximation of the ionicstrength conditions found throughout the body, including thegastrointestinal fluids, although concentrations higher than 0.1 M maybe employed to simulate fed conditions within the human GI tract.

Exemplary solutions of salts, acids, bases or combinations thereof,which exhibit the desired pH and ionic strength, include but are notlimited to phosphoric acid/phosphate salts+sodium, potassium and calciumsalts of chloride, acetic acid/acetate salts+sodium, potassium andcalcium salts of chloride, carbonic acid/bicarbonate salts+sodium,potassium and calcium salts of chloride, and citric acid/citratesalts+sodium, potassium and calcium salts of chloride.

In an exemplary method, aliquots of biorelevant aqueous media fromvessels containing the dosage form to be tested are removed atappropriate time points and the amount of redispersed active agent isquantitated by UV analysis at an appropriate wavelength using astandard. Other suitable assay methods such as chromatography can alsobe utilized in the methods of the invention. Confirmation of theparticle size of the active agent can be made using, e.g., a particlesize distribution analyzer. In cases where all components except theactive are completely water-soluble, the redispersibility process can bemonitored exclusively by particle size analysis. Conventional USPdissolution apparatus can also be utilized in the methods of theinvention.

Assay methods for nanoparticulate materials can be based on quantitationof all active agent in the sample after removal of larger material usingan appropriate filter technique. Alternatively, in situ spectroscopicdetection techniques sensitive to the size and/or concentration ofnanoparticulate active agents can be employed. A combination ofmultivariate analysis techniques and various forms of multi-wavelengthmolecular spectroscopy (ultraviolet (UV), visible (VIS), near infrared(NIR) and/or Raman resonance) can be used for simultaneous and rapidevaluation of both mean particle size and concentration of thenanoparticulate active agent.

In the methods of the invention, a dosage form of a nanoparticulateactive agent is expected to exhibit optimum in vivo performance whenupon reconstitution in a biorelevant aqueous medium, the dosage formredisperses such that the particle size distribution of the redispersednanoparticulate active agent particles resembles the distribution of theparticles prior to incorporation into the dosage form. For example, sucha prior particle size can be the particle size of the active agentpresent in a nanoparticulate active agent dispersion used to manufacturethe dosage form.

In the methods of the invention, a dosage form of a nanoparticulateactive agent is expected to exhibit optimum in vivo performance whenupon reconstitution in a biorelevant aqueous medium, the dosage formredisperses such that:

-   -   (a) if prior to incorporation into a dosage form the active        agent has an effective average particle size of less than about        2 microns, then following redispersibility 90% of the active        agent particles have a particle size of less than about 10        microns,    -   (b) if prior to incorporation into a dosage form the active        agent has an effective average particle size of less than about        1 micron, then following redispersibility 90% of the active        agent particles have a particle size of less than about 5        microns,    -   (c) if prior to incorporation into a dosage form the active        agent has an effective average particle size of less than about        800 nm, then following redispersibility 90% of the active agent        particles have a particle size of less than about 4 microns,    -   (d) if prior to incorporation into a dosage form the active        agent has an effective average particle size of less than about        600 nm, then following redispersibility 90% of the active agent        particles have a particle size of less than about 3 microns,    -   (e) if prior to incorporation into a dosage form the active        agent has an effective average particle size of less than about        400 nm, then following redispersibility 90% of the active agent        particles have a particle size of less than about 2 microns, and    -   (f) if prior to incorporation into a dosage form the active        agent has an effective average particle size of less than about        200 nm, then following redispersibility 90% of the active agent        particles have a particle size of less than about 1 micron.

Similarly, in the methods of the invention, a dosage form of amicroparticulate active agent is expected to exhibit optimum in vivoperformance when, upon reconstitution in a biorelevant aqueous medium,the dosage form redisperses such that the particle size distribution ofthe redispersed microparticulate active agent particles resembles thedistribution of the particles in the original microparticulate activeagent used to manufacture the dosage form.

The techniques of the present invention differ considerably fromconventional analytical methodologies for poorly water-soluble activeagents, which attempt to assess product quality by measuring the rateand extent of active agent dissolution, generally in the presence ofsurfactants or cosolvents. In contrast to prior methods of measuringdissolution, the methods of the present invention provide for directphysical measurement of an active agent's exposed surface area, i.e.,its redispersiblity. Moreover, this measurement is in the absence ofextraneous solubilizing agents that could otherwise decrease thesensitivity of the analytical test.

The in vitro redispersibility methods of the invention have beenimplemented successfully to select a dosage form for optimal in vivoperformance over the dosage form which would have been selected relyingon conventional dissolution techniques. Specifically, Example 1, below,shows the results of a comparison between the in vivo performance of aconventional solid dose microparticulate active agent, a firstgeneration solid dose nanoparticulate active agent, developed usingconventional dissolution methods, and a second generation solid dosenanoparticulate active agent, developed using the redispersibilitymethods of the invention (the active agent is the same for all threecompositions).

The data of Example 1, shown in FIG. 1, depict mean pharmacokineticprofiles in human volunteers following oral administration of single200-mg doses of two nanoparticulate active agent tablet formulations anda commercial capsule formulation (reference product). The primary goalof the study was to demonstrate an increased rate of active agentabsorption upon oral administration (i.e., faster onset of action).

The plasma level the plasma level at the 3-hour time point for thecommercial product was 495.04 ng/mL. Interpolating linearly between the0.25 and 0.50 hour time points for the nanoparticulate formulationindicates that a plasma level of 495 ng/mL is reached at 0.37 hours.This is about ⅛th of the time it takes to reach the same level with thecommercial formulation, or about 12% of the time it takes to reach thesame level with the commercial formulation. Thus, the results show thatthe “second generation” nanoparticulate active agent tablet formulation,developed using the redispersibility methodology of the invention,achieved a reduction in time to onset of approximately 88% (based upontime required to achieve maximum blood levels of ca. 500 ng/mL for thecommercial product) and further resulted in an unexpected increase inbioavailability of approximately 50%.

Surprisingly, the “first generation” nanoparticulate active agent tabletformulation, developed using conventional dissolution methodology,failed to improve time to onset or increase the bioavailability of theactive agent. Thus, particle size of the originally milled dispersionalone cannot predict the in vivo efficacy of a dosage form. Rather, invivo redispersibility of the dosage form is critical to in vivoefficacy.

Accordingly, the redispersibility methods of the invention are powerfultools for ensuring product quality throughout all stages of development,scale-up, and product transfer to commercial manufacturing sites.

Also provided by the invention are dosage forms selected by the methodof the invention, and methods of using such dosage forms. Such dosageforms include, but are not limited to, solid, semi-solid, or liquiddosage formulations, immediate release dosage forms, modified releasedosage forms, extended release dosage forms, delayed release dosageforms, pulsatile release dosage forms, controlled release dosage forms,fast melt (“waterless”) tablet formulations, dry powders, such as thosefor oral suspension or pulmonary or nasal administration,multiparticulates, sprinkles, tablets, capsules and related solidpresentations for oral administration, lyophilized formulations,sachets, lozenges, syrups, liquids for injection or oral delivery, etc.In sum, any dosage form can be designed using the methodology of theinvention, including but not limited to dosage forms intended for oral,pulmonary, nasal, parenteral, rectal, local, or buccal delivery.

The dosage forms identified using the method of the invention can beused to treat any human or animal in need.

A. Compositions to be Evaluated in a Method of the Invention

Any dosage form containing poorly soluble active ingredients can beevaluated according to the methods of the invention. The compositions tobe evaluated comprise at least one poorly water-soluble microparticulateactive agent, poorly water-soluble nanoparticulate active agent, or acombination thereof. By “poorly water-soluble” it is meant that theactive agent has a solubility in water of less than about 30 mg/ml, lessthan about 10 mg/mL, or less than about 1 mg/mL at ambient temperatureand pressure and at about pH 7.

As used in the present invention, a “nanoparticulate” active agent hasan effective average particle size of less than about 2 microns, and amicroparticulate active agent has an effective average particle size ofgreater than about 2 microns. Particle size is defined in more detailbelow.

Based upon overall physical properties and generalized appearance, adosage form of a nanoparticulate active agent is not readilydistinguishable from a traditional microparticulate active agent dosageform. Functionally, however, the performance of the nanoparticulateactive agent dosage form is enhanced considerably, due to the increasedrate of presentation of active agent to the absorbing surfaces of thegastrointestinal tract.

Proven therapeutic benefits of dosage forms of nanoparticulate activeagents include, but are not limited to: faster onset of action,increased bioavailability, reduced fed/fasted variable absorption (i.e.,“food effect”), and improved dose proportionality. Other potentialadvantages of nanoparticulate active agent technology relevant to thedevelopment and manufacture of pharmaceutical dosage forms include:improved chemical stability of the active agent, improved physicalstability and performance stability of the dosage form, enhancedappearance (i.e., elegant, compact presentation), improvedprocessability and reduced impact of lot-to-lot variability in activeagent, rapid development, and predictable performance throughout productdevelopment, scale-up, and transfer to commercial manufacturing site.

1. Active Agents

The active agent may be present either substantially in the form of oneoptically pure enantiomer or as a mixture, racemic or otherwise, ofenantiomers. In addition, the active agent exists as a discrete,crystalline phase, as an amorphous phase, a semi-crystalline phase, asemi-amorphous phase, or a combination thereof.

Exemplary active agents can be therapeutic or diagnostic agents,collectively referred to as “drugs”. A therapeutic agent can be apharmaceutical agent, including biologics such as proteins, peptides,and nucleotides, or-a diagnostic agent, such as a contrast agent,including x-ray contrast agents.

The active agent can be selected from a variety of known classes ofdrugs, including, for example, COX-2 inhibitors, retinoids, anticanceragents, NSAIDS, proteins, peptides, nucleotides, anti-obesity drugs,nutraceuticals, corticosteroids, elastase inhibitors, analgesics,anti-fungals, oncology therapies, anti-emetics, analgesics,cardiovascular agents, anti-inflammatory agents, anthelmintics,anti-arrhythmic agents, antibiotics (including penicillins),anticoagulants, antidepressants, antidiabetic agents, antiepileptics,antihistamines, antihypertensive agents, antimuscarinic agents,antimycobacterial agents, antineoplastic agents, immunosuppressants,antithyroid agents, antiviral agents, anxiolytics, sedatives(e.g.,hypnotics and neuroleptics), astringents, beta-adrenoceptorblocking agents, blood products and substitutes, cardiac inotropicagents, contrast media, corticosteroids, cough suppressants(expectorants and mucolytics), diagnostic agents, diagnostic imagingagents, diuretics, dopaminergics (antiparkinsonian agents),haemostatics, immunological agents, lipid regulating agents, musclerelaxants, parasympathomimetics, parathyroid calcitonin andbiphosphonates, prostaglandins, radio-pharmaceuticals, sex hormones(including steroids), anti-allergic agents, stimulants and anoretics,sympathomimetics, thyroid agents, vasodilators, xanthines, alpha-hydroxyformulations, cystic-fibrosis therapies, asthma therapies, emphysematherapies, respiratory distress syndrome therapies, chronic bronchitistherapies, chronic obstructive pulmonary disease therapies,organ-transplant rejection therapies, therapies for tuberculosis andother infections of the lung, and respiratory illness therapiesassociated with acquired immune deficiency syndrome.

Exemplary nutraceuticals and dietary supplements are disclosed, forexample, in Roberts et al., Nutraceuticals: The Complete Encyclopedia ofSupplements, Herbs, Vitamins, and Healing Foods (American NutraceuticalAssociation, 2001), which is specifically incorporated by reference. Anutraceutical or dietary supplement, also known as phytochemicals orfunctional foods, is generally any one of a class of dietarysupplements, vitamins, minerals, herbs, or healing foods that havemedical or pharmaceutical effects on the body. Exemplary nutraceuticalsor dietary supplements include, but are not limited to, lutein, folicacid, fatty acids (e.g., DHA and ARA), fruit and vegetable extracts,vitamin and mineral supplements, phosphatidylserine, lipoic acid,melatonin, glucosamine/chondroitin, Aloe Vera, Guggul, glutamine, aminoacids (e.g., iso-leucine, leucine, lysine, methionine, phenylanine,threonine, tryptophan, and valine), green tea, lycopene, whole foods,food additives, herbs, phytonutrients, antioxidants, flavonoidconstituents of fruits, evening primrose oil, flax seeds, fish andmarine animal oils, and probiotics. Nutraceuticals and dietarysupplements also include bio-engineered foods genetically engineered tohave a desired property, also known as “pharmafoods.”

The active agents are commercially available and/or can be prepared bytechniques known in the art.

2. Surface Stabilizers for Nanoparticulate Active Agents

If the active agent has a nanoparticulate particle size prior toincorporation into a dosage form, with “nanoparticulate” being definedas an effective average particle size of less than about 2 microns, thenthe active agent generally will have at least one surface stabilizeradsorbed on the surface of the active agent.

Surface stabilizers useful herein physically adhere on the surface ofthe nanoparticulate active agent but do not chemically react with theactive agent particles or itself. Individually adsorbed molecules of thesurface stabilizer are essentially free of intermolecularcross-linkages.

Exemplary useful surface stabilizers include, but are not limited to,known organic and inorganic pharmaceutical excipients. Such excipientsinclude various polymers, low molecular weight oligomers, naturalproducts, and surfactants. Preferred surface stabilizers includenonionic and ionic surfactants, including anionic and cationicsurfactants. Combinations of more than one surface stabilizer can beused in the invention.

Representative examples of surface stabilizers include hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, randomcopolymers of vinyl pyrrolidone and vinyl acetate, sodium laurylsulfate, dioctylsulfosuccinate, gelatin, casein, lecithin(phosphatides), dextran, gum acacia, cholesterol, tragacanth, stearicacid, benzalkonium chloride, calcium stearate, glycerol monostearate,cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters,polyoxyethylene alkyl ethers (e.g., macrogol ethers such as cetomacrogol1000), polyoxyethylene castor oil derivatives, polyoxyethylene sorbitanfatty acid esters (e.g., the commercially available Tweens® such ase.g., Tween 20® and Tween 80® (ICI Speciality Chemicals)); polyethyleneglycols (e.g., Carbowaxs 3550® and 934® (Union Carbide)),polyoxyethylene stearates, colloidal silicon dioxide, phosphates,carboxymethylcellulose calcium, carboxymethylcellulose sodium,methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulosephthalate, noncrystalline cellulose, magnesium aluminium silicate,triethanolamine, polyvinyl alcohol (PVA),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol, superione, and triton),poloxamers (e.g., Pluronics F68® and F108®, which are block copolymersof ethylene oxide and propylene oxide); poloxamines (e.g., Tetronic908®, also known as Poloxamine 908®, which is a tetrafunctional blockcopolymer derived from sequential addition of propylene oxide andethylene oxide to ethylenediamine (BASF Wyandotte Corporation,Parsippany, N.J.)); Tetronic 1508® (T-1508) (BASF WyandotteCorporation), Tritons X-200®, which is an alkyl aryl polyether sulfonate(Dow); Crodestas F-110®, which is a mixture of sucrose stearate andsucrose distearate (Croda Inc.); p-isononylphenoxypoly-(glycidol), alsoknown as Olin-IOG® or Surfactant 10-G® (Olin Chemicals, Stamford,Conn.); Crodestas SL-40® (Croda, Inc.); and SA9OHCO, which isC₁₈H₃₇CH₂C(O)N(CH₃)—CH₂(CHOH)₄(CH₂OH)₂ (Eastman Kodak Co.);decanoyl-N-methylglucamide; n-decyl β-D-glucopyranoside; n-decylβ-maltopyranoside; n-dodecyl β-D-glucopyranoside; n-dodecylβ-D-maltoside; heptanoyl-N-methylglucamide;n-heptyl-β-D-glucopyranoside; n-heptyl β-D-thioglucoside; n-hexylβ-D-glucopyranoside; nonanoyl-N-methylglucamide; n-noylβ-D-glucopyranoside; octanoyl-N-methylglucamide;n-octyl-β-D-glucopyranoside; octyl β-D-thioglucopyranoside;PEG-phospholipid, PEG-cholesterol, PEG-cholesterol derivative,PEG-vitamin A, PEG-vitamin E, lysozyme, and the like.

Examples of useful cationic surface stabilizers include, but are notlimited to, polymers, biopolymers, polysaccharides, cellulosics,alginates, phospholipids, and nonpolymeric compounds, such aszwitterionic stabilizers, poly-n-methylpyridinium, anthryul pyridiniumchloride, cationic phospholipids, chitosan, polylysine,polyvinylimidazole, polybrene, polymethylmethacrylatetrimethylammoniumbromide bromide (PMMTMABr), hexyldesyltrimethylammoniumbromide (HDMAB), and polyvinylpyrrolidone-2-dimethylaminoethylmethacrylate dimethyl sulfate.

Other useful cationic stabilizers include, but are not limited to,cationic lipids, sulfonium, phosphonium, and quarternary ammoniumcompounds, such as stearyltrimethylammonium chloride,benzyl-di(2-chloroethyl)ethylammonium bromide, coconut trimethylammonium chloride or bromide, coconut methyl dihydroxyethyl ammoniumchloride or bromide, decyl triethyl ammonium chloride, decyl dimethylhydroxyethyl ammonium chloride or bromide, C₁₂₋₁₅ dimethyl hydroxyethylammonium chloride or bromide, coconut dimethyl hydroxyethyl ammoniumchloride or bromide, myristyl trimethyl ammonium methyl sulphate, lauryldimethyl benzyl ammonium chloride or bromide, lauryl dimethyl(ethenoxy)₄ ammonium chloride or bromide, N-alkyl (C₁₂₋₁₈)dimethylbenzyl ammonium chloride, N-alkyl (C₁₄₋₁₈) dimethyl-benzylammonium chloride, N-tetradecylidmethylbenzyl ammonium chloridemonohydrate, dimethyl didecyl ammonium chloride, N-alkyl and (C₁₂₋₁₄)dimethyl 1-napthylmethyl ammonium chloride, trimethylammonium halide,alkyl-trimethylammonium salts and dialkyl-dimethylammonium salts, lauryltrimethyl ammonium chloride, ethoxylated alkyamidoalkyldialkylammoniumsalt and/or an ethoxylated trialkyl ammonium salt, dialkylbenzenedialkylammonium chloride, N-didecyldimethyl ammonium chloride,N-tetradecyldimethylbenzyl ammonium, chloride monohydrate,N-alkyl(C₁₂₋₁₄) dimethyl 1-naphthylmethyl ammonium chloride anddodecyldimethylbenzyl ammonium chloride, dialkyl benzenealkyl ammoniumchloride, lauryl trimethyl ammonium chloride, alkylbenzyl methylammonium chloride, alkyl benzyl dimethyl ammonium bromide, C₁₂, C₁₅, C₁₇trimethyl ammonium bromides, dodecylbenzyl triethyl ammonium chloride,poly-diallyldimethylammonium chloride (DADMAC), dimethyl ammoniumchlorides, alkyldimethylammonium halogenides, tricetyl methyl ammoniumchloride, decyltrimethylammonium bromide, dodecyltriethylammoniumbromide, tetradecyltrimethylammonium bromide, methyl trioctylammoniumchloride (ALIQUAT 336™), POLYQUAT 10™, tetrabutylammonium bromide,benzyl trimethylammonium bromide, choline esters (such as choline estersof fatty acids), benzalkonium chloride, stearalkonium chloride compounds(such as stearyltrimonium chloride and Di-stearyldimonium chloride),cetyl pyridinium bromide or chloride, halide salts of quaternizedpolyoxyethylalkylamines, MIRAPOL™ and ALKAQUAT™ (Alkaril ChemicalCompany), alkyl pyridinium salts; amines, such as alkylamines,dialkylamines, alkanolamines, polyethylenepolyamines,N,N-dialkylaminoalkyl acrylates, and vinyl pyridine, amine salts, suchas lauryl amine acetate, stearyl amine acetate, alkylpyridinium salt,and alkylimidazolium salt, and amine oxides; imide azolinium salts;protonated quaternary acrylamides; methylated quaternary polymers, suchas poly[diallyl dimethylammonium chloride] and poly-[N-methyl vinylpyridinium chloride]; and cationic guar.

Such exemplary cationic surface stabilizers and other useful cationicsurface stabilizers are described in J. Cross and E. Singer, CationicSurfactants: Analytical and Biological Evaluation (Marcel Dekker, 1994);P. and D. Rubingh (Editor), Cationic Surfactants: Physical Chemistry(Marcel Dekker, 1991); and J. Richmond, Cationic Surfactants: OrganicChemistry, (Marcel Dekker, 1990).

Particularly preferred nonpolymeric primary stabilizers are anynonpolymeric compound, such benzalkonium chloride, a carbonium compound,a phosphonium compound, an oxonium compound, a halonium compound, acationic organometallic compound, a quarternary phosphorous compound, apyridinium compound, an anilinium compound, an ammonium compound, ahydroxylammonium compound, a primary ammonium compound, a secondaryammonium compound, a tertiary ammonium compound, and quarternaryammonium compounds of the formula NR₁R₂R₃R₄ ⁽⁺⁾. For compounds of theformula NR₁R₂R₃R₄ ⁽⁺⁾:

-   (i) none of R₁–R₄ are CH₃;-   (ii) one of R₁–R₄ is CH₃;-   (iii) three of R₁–R₄ are CH₃;-   (iv) all of R₁–R₄ are CH₃;-   (v) two of R₁–R₄ are CH₃, one of R₁–R₄ is C₆H₅CH₂, and one of R₁–R₄    is an alkyl chain of seven carbon atoms or less;-   (vi) two of R₁–R₄ are CH₃, one of R₁–R₄ is C₆H₅CH₂, and one of R₁–R₄    is an alkyl chain of nineteen carbon atoms or more;-   (vii) two of R₁–R₄ are CH₃ and one of R₁–R₄ is the group    C₆H₅(CH₂)_(n), where n>1;-   (viii) two of R₁–R₄ are CH₃, one of R₁–R₄ is C₆H₅CH₂, and one of    R₁–R₄ comprises at least one heteroatom;-   (ix) two of R₁–R₄ are CH₃, one of R₁–R₄ is C₆H₅CH₂, and one of R₁–R₄    comprises at least one halogen;-   (x) two of R₁–R₄ are CH₃, one of R₁–R₄ is C₆H₅CH₂, and one of R₁–R₄    comprises at least one cyclic fragment;-   (xi) two of R₁–R₄ are CH₃ and one of R₁–R₄ is a phenyl ring; or-   (xii) two of R₁–R₄ are CH₃ and two of R₁–R₄ are purely aliphatic    fragments.

Such compounds include, but are not limited to, behenalkonium chloride,benzethonium chloride, cetylpyridinium chloride, behentrimoniumchloride, lauralkonium chloride, cetalkonium chloride, cetrimoniumbromide, cetrimonium chloride, cethylamine hydrofluoride,chlorallylmethenamine chloride (Quaternium-15), distearyldimoniumchloride (Quatemium-5), dodecyl dimethyl ethylbenzyl ammoniumchloride(Quaternium-14), Quaternium-22, Quaternium-26, Quaternium-18hectorite, dimethylaminoethylchloride hydrochloride, cysteinehydrochloride, diethanolammonium POE (10) oletyl ether phosphate,diethanolammonium POE (3)oleyl ether phosphate, tallow alkoniumchloride, dimethyl dioctadecylammoniumbentonite, stearalkonium chloride,domiphen bromide, denatonium benzoate, myristalkonium chloride,laurtrimonium chloride, ethylenediamine dihydrochloride, guanidinehydrochloride, pyridoxine HCl, iofetamine hydrochloride, megluminehydrochloride, methylbenzethonium chloride, myrtrimonium bromide,oleyltrimonium chloride, polyquaternium-1, procainehydrochloride,cocobetaine, stearalkonium bentonite, stearalkoniumhectonite, stearyltrihydroxyethyl propylenediamine dihydrofluoride, tallowtrimoniumchloride, and hexadecyltrimethyl ammonium bromide.

Most of these surface stabilizers are known pharmaceutical excipientsand are described in detail in the Handbook of PharmaceuticalExcipients, published jointly by the American Pharmaceutical Associationand The Pharmaceutical Society of Great Britain (The PharmaceuticalPress, 2000), specifically incorporated by reference. The surfacestabilizers are commercially available and/or can be prepared bytechniques known in the art.

3. Microparticulate and Nanoparticulate Particle Size of the PoorlyWater-Soluble Active Agent

As used herein, particle size is determined on the basis of the weightaverage particle size as measured by conventional particle sizemeasuring techniques well known to those skilled in the art. Suchtechniques include, for example, sedimentation field flow fractionation,photon correlation spectroscopy, light scattering, and diskcentrifugation.

By “an effective average particle size of less than about 2 microns” itis meant that the arithmetic mean of the weight distribution (the weightfraction as a function of particle size) is less than about 2 micronswhen measured by the above techniques.

Similarly, by “an effective average particle size of greater than about2 microns” it is meant that at the arithmetic mean of the weightdistribution of the active agent particles is greater than about 2microns when measured by the above techniques.

If the active agent has a nanoparticulate particle size, then at leastabout 50%, about 70%, about 90%, or about 95% of the active agentparticles can have an average particle size of less than the effectiveaverage, i.e., less than about 2 microns.

In addition, in other embodiments of the invention, the effectiveaverage particle size of the nanoparticulate active agent particles canbe less than about 1500 nm, less than about 1000 nm, less than about 900nm, less than about 800 nm, less than about 700 nm, less than about 600nm, less than about 500 nm, less than about 400 nm, less than about 300nm, less than about 250 nm, less than about 200 nm, less than about 100nm, less than about 75 nm, or less than about 50 nm.

If the active agent has a microparticulate particle size, the effectiveaverage particle size of the microparticulate active agent compositioncan be greater than about 2 microns, greater than about 5 microns,greater than about 10 microns, greater than about 15 microns, andgreater than about 20 microns.

4. Concentration of Nanoparticulate Active Agent and Surface Stabilizer

If the active agent is in a nanoparticulate particle size, then theactive agent has one or more surface stabilizers adsorbed onto thesurface of the agent. The relative amount of active agent and one ormore surface stabilizers can vary widely. The optimal amount of thesurface stabilizer(s) can depend, for example, upon the particularactive agent selected, the equivalent hydrophilic lipophilic balance(HLB) of the active agent, the melting point, cloud point, and watersolubility of the surface stabilizer, and the surface tension of watersolutions of the stabilizer, etc.

The concentration of at least one active agent can vary from about 99.5%to about 0.001%, from about 95% to about 0.1%, or from about 90% toabout 0.5%, by weight, based on the total combined weight of the atleast one active agent and at least one surface stabilizer, notincluding other excipients.

The concentration of at least one surface stabilizer can vary from about0.5% to about 99.999%, from about 5% to about 99.9%, and from about 10%to about 99.5%, by weight, based on the total combined dry weight of atleast one active agent and at least one surface stabilizer, notincluding other excipients.

B. Methods of Making Nanoparticulate Formulations

Nanoparticulate active agent compositions can be made using methodsknown in the art such as, for example, milling, homogenization, andprecipitation techniques.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including a U.S. patent, are specificallyincorporated by reference.

In the examples that follow, the value for D50 is the particle sizebelow which 50% of the active agent particles fall. Similarly, D90 isthe particle size below which 90% of the active agent particles fall.

EXAMPLE 1

The purpose of this example was to compare the in vivo performance of aconventional solid dose microparticulate active agent, a firstgeneration solid dose nanoparticulate active agent, and a secondgeneration solid dose nanoparticulate active agent developed using theredispersibility methods of the invention. The active agent is poorlywater-soluble.

Human volunteers were orally administered a single 200-mg dose of aconventional solid dose microparticulate active agent, a firstgeneration solid dose nanoparticulate active agent, and a secondgeneration solid dose nanoparticulate active agent. FIG. 1 shows themean pharmacokinetic profiles of each dosage form followingadministration. Greater plasma levels of active agent correspond togreater bioavailability of administered active agent.

Surprisingly, the first generation solid dose nanoparticulate activeagent dosage form exhibited minimal, if any, increased bioavailabilityor time to onset over the conventional microparticulate dosage formwhile the in vitro dissolution data indicated that a superiorperformance over the conventional dosage form should be expected.

Only the “second generation” nanoparticulate active agent tabletformulation, developed using the redispersibility methodology of theinvention, achieved a reduction in time to onset of approximately 90%(based upon time required to achieve maximum blood levels of ca. 500ng/mL for the commercial product) and further resulted in an unexpectedincrease in bioavailability of approximately 50%.

The results of this example demonstrate that active agent particle sizein the solid dose form alone will not determine the in vivoeffectiveness of an orally administered solid dosage form. Rather,redispersibility of the component active agent is critical to in vivosuccess. Thus, the ability to demonstrate redispersibility greatly aidsin the solid dosage form design. That is the design of a solid dose formpredictive of optimum in vivo performance characteristics.

EXAMPLE 2

The purpose of this example was to evaluate the redispersibilityproperties of a solid dose nanoparticulate ketoprofen composition in anelectrolyte solution. Ketoprofen, also known as m-benzoylhydratopic acidis a nonsteroidal anti-inflammatory analgesic. The drug is poorlywater-soluble.

A ketoprofen nanoparticulate dispersion was prepared having 5%ketoprofen, 1.% polyvinyl pyrrolidone (PVP) K29/32, and 0.2% dioctylsodium sulfosuccinate (DOSS). The dispersion was prepared using aDyno®—Mill (Type: KDL; Mfg.: Willy A Bachofen AG, Basel Switzerland)equipped with a 150 cc batch chamber using a 500 μm milling media oftype Polymill500® for 2 hrs at 10° C.

The ketoprofen nanoparticulate dispersion (ketoprofen NCD) was thenspray dried with mannitol, with a drug to mannitol ratio of 1:1 using aBüchi Mini Spray Dryer B-191 (Büchi, Switzerland). The redispersibilityproperties of the spray dried ketoprofen composition in water are shownbelow in Table 1.

TABLE 1 Redispersibility Properties of a Solid Dose NanoparticulateKetoprofen Composition in Water Mean Mean D₅₀ D₅₀ D₉₀ D₉₀ Time (no (1min. (1 min. (no (1 min. (no (days) sonication) sonication) sonication)sonication) sonication) sonication) 0 118 121 105 107 192 198 1 152 163144 155 219 233 All measurements are in nanometers (nm).

The results of the redispersibility test show excellent redispersibilityof the spray dried nanoparticulate composition.

The redispersibility properties of the same spray dried ketoprofencomposition were then tested in electrolyte solutions, which mimic theconditions found in the human gastrointestinal tract. The results ofthese tests are shown in Table 2, below.

TABLE 2 Redispersibility Properties of a Solid Dose NanoparticulateKetoprofen Composition in Electrolyte Solutions 1 min. 1 min.Electrolyte no sonic. No. sonic. No. sonic. sonic. sonic. No. sonic.Conc. (M) Type Mean Small % Large % Mean Small % Large % 0 — 172 100 0182 100 0 0.001 HCl 535 97 3 166 100 0 0.01 HCl 176 100 0 188 100 0 0.1HCl 17756 2 98 5908 8 92 0.001 NaCl 178 100 0 191 100 0 0.01 NaCl 151100 0 163 100 0 0.1 NaCl 186 100 0 204 100 0 All particle sizes are innanometers (nm).

“Small” particles are defined as those below 1 micron (1000 nm) and“large” particles are those above 1 micron. Electrolyte concentrationsof 0.001 M HCl, 0.01 M HCl, and 0.1 M HCl correspond to pH 3, pH 2, andpH 1, respectively. In the stomach, the pH ranges from slightly lessthan 2 (but typically greater than 1) up to 4 or 5. In the smallintestine the pH can range from 4 to 6, and in the colon it can rangefrom 6 to 8. Thus, a 0.01 M HCl concentration simulates typical acidicconditions found in the stomach. 0.1 M NaCl simulates the electrolyteconcentration found throughout the body, including the intestine.

The results show that under acidic to neutral pH conditions, thenanoparticulate ketoprofen solid dose composition showed excellentredispersibility properties, with 100% of the nanoparticulate particleshaving a redispersed particle size of less than 1 micron. In addition,under all but the most acidic conditions of 0.1 M HCl (which are nottypically representative of human gastric pH), the nanoparticulateketoprofen solid dose composition showed excellent redispersibilityproperties, with almost 100% of the nanoparticulate particles having aredispersed particle size of less than 1 micron.

EXAMPLE 3

The purpose of this example was to evaluate the redispersibilityproperties of a solid dose nanoparticulate MAP kinase inhibitorcomposition in electrolyte solutions.

5% (w/w) of Compound A, a poorly water-soluble MAP kinase inhibitor, 1%Plasdone® S630, and 0.2% DOSS were milled using a Dyno®—Mill (Type: KDL;Mfg.: Willy A Bachofen AG, Basel, Switzerland) equipped with a 150 ccbatch chamber using a 500 μm milling media of type Polymill500® for 3hrs at 10° C. Plasdone® S630 is a random copolymer of vinyl acetate andvinyl pyrrolidone.

The nanoparticulate MAP kinase inhibitor dispersion (NCD) was then spraydried at a drug to mannitol ratio of 1:1 using a Büchi Mini Spray DryerB-191 (Büchi, Switzerland). The redispersibility properties of the spraydried MAP kinase inhibitor in electrolyte solutions are shown below inTable 3 and in FIG. 2. A Horiba LA910 particle sizer was used to measureparticle size. “Small” particles were defined as those below 1 micronand “large” particles were defined as those above 1 micron.

TABLE 3 Redispersibility Properties of a Solid Dose Nanoparticulate MAPKinase Inhibitor Composition in Electrolyte Solutions 1 min. 1 min.Electrolyte no sonic. No. sonic. No. sonic. sonic. sonic. No. sonic.Conc. (M) Type Mean Small % Large % Mean Small % Large % 0 — 99 100 0 99100 0 0.001 HCl 100 100 0 100 100 0 0.01 HCl 105 100 0 106 100 0 0.1 HCl4708 23 77 1901 52 48 0.001 NaCl 103 100 0 103 100 0 0.01 NaCl 101 100 0101 100 0 0.1 NaCl 105 100 0 105 100 0 All particle sizes are innanometers (nm).

The results show that the solid dose nanoparticulate MAP kinaseinhibitor composition showed excellent redispersibility in all testedelectrolyte media representative of in vivo conditions. Even at a higheracid concentration of 0.1 M HCl, the composition showed over 50% of thedrug particles of the composition having a small particle size following1 minute of sonication.

EXAMPLE 4

The purpose of this example was to evaluate the redispersibilityproperties of a solid dose nanoparticulate angiogenesis inhibitorcomposition in water and in electrolyte solutions.

Nanocrystalline dispersions (NCD) of a poorly water-soluble angiogenesisinhibitor, Compound C, were made by milling the ingredients shown foreach composition in Table 4. Samples A and B were milled on a NetzchMill (Netzsch Inc., Exton, Pa.), having a LMZ 2L chamber, for 11 hrs.500 micron PolyMill media was used. Processing temperatures ranged from11.6° C. to 27.4° C. Samples C-E were milled on a Dyno® Mill, having a150 cc chamber, at a temperature of 10° C. for 3 hours, also using 500micron PolyMill media.

Following milling, the additives listed in Table 4 were added to thenanoparticulate dispersion until dissolved, followed by spraying of thedispersion over a fluidized mannitol excipient, also provided in Table4, to form a solid dose composition. A Glatt GPCG-1 fluid bed processor(Glatt Air Technologies, Inc., Ramsey, N.J.) was used for this process.

TABLE 4 Spray Granulated Nanoparticulate Angiogenesis InhibitorCompositions Particle Size of Nano- Sample Formula crystal Dispersion(nm) Additives Fluidized Mannitol A 15% Drug + 3.75% mean 105 nm; Drug:mannitol Pearlitol ® SD200 PVP K29/32 and D₉₀ of 167 nm ratio of 1:0.750.15% sodium lauryl sulfate (SLS) B 15% Drug + 3.75% mean 105 nm; Drug:mannitol Pearlitol ® SD200 PVP K29/32 and D₉₀ of 167 nm ratio of 1:0.750.15% SLS C 15% Drug + 3.75% mean of 101 nm; Drug: mannitol Mannitol 35PVP K29/32, 0.15% D₉₀ of 165 nm ratio of 1:0.75 SLS, and 0.1% sodiumascorbate D 15% Drug + 3.75% mean of 101 nm; Drug: mannitol Mannitol 35PVP K29/32, 0.15% D₉₀ of 165 nm ratio of 1:0.75 SLS, and 0.1% sodiumascorbate E 15% Drug + 3.75% mean of 101 nm; Drug: mannitol Mannitol 35PVP K29/32, 0.15% D₉₀ of 165 nm ratio of 1:0.75 SLS, and 0.1% andstabilizer sodium ascorbate DOSS ratio of 1:0.2

Each composition A-E, comprising drug/excipient granules, was thenmilled to a uniform particle size in a Quadro Comill (Model 193; alsocalled a cone mill, which comprises fixed stationary screens and arotating impeller), to produce Compositions A2-E2. The milling processcomprised passing the powder through the mill (one pass through, about2–5 minutes).

The redispersibility, in water and various electrolyte solutions, wasthen measured for the solid dose nanoparticulate angiogenesis inhibitorcompositions, both Compositions A-E (unmilled) and A2-E2 (milled), asshown in Table 5.

TABLE 5 Redispersibility of Solid Dose Nanoparticulate AngiogenesisInhibitor Compositions (Milled and Unmilled Granulate Compositions) inWater and Electrolyte Solutions No Sonication 1 Min. Sonication Redisp.Mean D90 % Under Mean D90 % Under Composition Media (nm) (nm) 1000 nm(nm) (nm) 1000 nm A water 5265 11776 26.2 1440 4717 70.8 (unmilled) 0.01M HCl 12160 27244 9.4 3034 6997 36.1 0.01 M NaCl 7487 15324 11.6 22746504 57.6 A2 Water 5777 12463 23.0 2538 7547 62.9 (milled) 0.01 M HCl58519 236602 5.3 3573 7929 30 0.01 M NaCl 8341 17698 11 1975 5366 54.9 BWater 8222 18365 18.5 4368 9033 51.5 (unmilled) 0.01 M HCl 83643 2645454.8 4238 9458 26.3 0.01 M NaCl 14863 33139 8 2579 6561 45.8 B2 Water18897 55523 14.2 2691 7294 50 (milled) 0.01 M HCl 44037 103747 4.1 516111771 22.4 0.01 M NaCl 13514 29820 6.8 2547 6163 42.1 C Water 3124 808846.9 422 645 93.4 (unmilled) 0.01 M HCl 6713 14117 16.6 2471 6285 470.01 M NaCl 4103 9426 30.6 904 3006 80.4 C2 Water 3150 8427 49 1071 360283.6 (milled) 0.01 M HCl 8728 19180 17.1 3039 7626 43.3 0.01 M NaCl 45449896 25.5 1278 4345 75 D Water 3094 7865 44.8 342 569 97.3 (unmilled)0.01 M HCl 9630 21697 14.8 2762 7043 45.3 0.01 M NaCl 4295 8561 20.61475 5034 73.6 D2 Water 2162 5885 54.4 295 488 98.7 (milled) 0.01 M HCl8885 20181 16.9 1982 5087 51.7 0.01 M NaCl 4410 8710 19 1066 3420 75.9 EWater 2186 7520 69.9 384 614 98.3 (unmilled) 0.01 M HCl 2161 7812 73.4297 492 99 0.01 M NaCl 2544 8755 68.1 357 588 98.5 E Water 2711 914166.6 436 672 93.6 (milled) 0.01 M HCl 2014 7608 75.9 291 483 99.1 0.01 MNaCl 2203 8075 74.1 292 484 99

The redispersibility results first show that redispersibility in waterdoes not predict redispersibility in an electrolyte solution.

Second, the redispersibility results show only one sample, Sample E,showed good redispersibility in electrolyte media, with aredispersibility of 99.1% in 0.01 M HCl and 99% in 0.01 M NaCl. Incontrast, Samples A-D showed redispersibility in 0.01 M HCl of from22.4% (Sample B2) to 51.7% (Sample D2), and a redispersibility in 0.01 MHCl of from to 42.1% (Sample B2) to 80.4% (Sample C). The results aredramatic as the only difference between Sample E and Samples C and D wasthe presence (Sample E) or absence (Samples C and D) of DOSS.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of this invention provided they come withinthe scope of the appended claims and their equivalents.

1. An in vitro method for evaluating a dosage form, comprising: (a)redispersing a dosage form, comprising at least one poorly water-solubleactive agent, in at least one biorelevant aqueous medium, wherein priorto incorporation into the dosage form the active agent has an effectiveaverage particle size of less than about 2 microns; (b) measuring theparticle size of the redispersed poorly water-soluble active agent; and(c) determining if the level of redispersibility is sufficient for invivo effectiveness of the dosage form, wherein the level ofredispersibility is sufficient for in vivo effectiveness of the dosageform if the dosage form redisperses such that at least 90% of the activeagent particles have a particle size of less than about 10 microns. 2.The method of claim 1, wherein the dosage form is selected from thegroup consisting of solid dosage forms, liquid dosage forms, semi-liquiddosage forms dry powders, multiparticulates, sprinkles, tablets,capsules, lyophilized formulations, sachets, lozenges, syrups, liquidsfor injection, and liquids for oral delivery.
 3. The method of claim 1,wherein the dosage form is selected from the group consisting of dosageforms intended for oral, pulmonary, nasal, parenteral, rectal, local,and buccal delivery.
 4. The method of claim 1, wherein prior toincorporation into the dosage form the active agent has an effectiveaverage particle size selected from the group consisting of less thanabout 1500 nm, less than about 1000 nm, less than about 900 nm, lessthan about 800 nm, less than about 700 nm, less than about 600 nm, lessthan about 500 nm, less than about 400 nm, less than about 300 nm, lessthan about 250 nm, less than about 200 nm, less than about 100 nm, lessthan about 75 nm, and less than about 50 nm.
 5. The method of claim 1,wherein the biorelevant aqueous media is selected from the groupconsisting of electrolyte solutions of strong acids, strong bases, weakacids, weak bases, and salts thereof, and mixtures of strong acids,strong bases, weak acids, weak bases, and salts thereof.
 6. The methodof claim 5, wherein the electrolyte solution is selected from the groupconsisting of an HCl solution having a concentration from about 0.00 1to about 0.1 M, an NaCl solution having a concentration from about 0.001 to about 0.2 M, and mixtures thereof.
 7. The method of claim 5,wherein the electrolyte solution is selected from the group consistingof about 0.1 M HCl or less, about 0.01 M HCl or less, about 0.001 M HClor less, about 0.2 M NaCl or less, about 0.01 M NaCl or less, about 0.001 M NaCl or less, and mixtures thereof.
 8. The method of claim 5,wherein the electrolyte solution is selected from the group consistingof 0.01 M HCl and/or 0.1 M NaCl.
 9. The method of claim 1, wherein thedosage form additionally comprises at least one surface stabilizeradsorbed to the surface of the active agent.
 10. The method of claim 9,wherein the at least one active agent is present in an amount selectedfrom the group consisting of from about 99.5% to about 0.001%, fromabout 95% to about 0.1%, and from about 90% to about 0.5%, by weight,based on the total combined weight of the at least one active agent andat least one surface stabilizer, not including other excipients.
 11. Themethod of claim 9, wherein the at least one surface stabilizer ispresent in an amount selected from the group consisting of from about0.5% to about 99.999%, from about 5% to about 99.9%, and from about 10%to about 99.5%, by weight, based on the total combined dry weight of theat least one active agent and at least one surface stabilizer, notincluding other excipients.
 12. The method of claim 9, wherein the atleast one surface stabilizer is selected from the group consisting of anon-ionic surface stabilizer, a cationic surface stabilizer, and ananionic surface stabilizer.
 13. The method of claim 1, wherein theactive agent is selected from the group consisting of a crystallinephase active agent, a semi-crystalline phase active agent, an amorphousphase active agent, a semi-amorphous phase active agent, and a mixturethereof.
 14. The method of claim 1, wherein the at least one activeagent is selected from the group consisting of COX-2 inhibitors,anticancer agents, NSAIDS, proteins, peptides, nutraceuticals,anti-obesity agents, corticosteroids, elastase inhibitors, analgesics,anti-fungals, oncology therapies, anti-emetics, analgesics,cardiovascular agents, anti-inflammatory agents, anthelmintics,anti-arrhythmic agents, antibiotics, anticoagulants, antidepressants,antidiabetic agents, antiepileptics, antihistamines, antihypertensiveagents, antimuscarinic agents, antimycobacterial agents, antineoplasticagents, immunosuppressants, antithyroid agents, antiviral agents,anxiolytics, sedatives, astringents, beta-adrenoceptor blocking agents,blood products and substitutes, cardiac inotropic agents, contrastmedia, cough suppressants, diagnostic agents, diagnostic imaging agents,diuretics, dopaminergics, haemostatics, immunological agents, lipidregulating agents, muscle relaxants, parasympathomimetics, parathyroidcalcitonin and biphosphonates, prostaglandins, radio-pharmaceuticals,sex hormones, anti-allergic agents, stimulants and anoretics,sympathomimetics, thyroid agents, vasodilators, xanthines, acnemedication, alpha-hydroxy formulations, cystic-fibrosis therapies,asthma therapies, emphysema therapies, respiratory distress syndrometherapies, chronic bronchitis therapies, chronic obstructive pulmonarydisease therapies, organ-transplant rejection therapies, therapies fortuberculosis and other infections of the lung, and respiratory illnesstherapies associated with acquired immune deficiency syndrome.
 15. Themethod of claim 1, wherein the level of redispersibility is sufficientfor in vivo effectiveness of the dosage form if the active agent has aneffective average particle size prior to incorporation into the dosageform of less than about 1 micron, and upon reconstitution in mediarepresentative of human physiological conditions the dosage formredisperses such that 90% of the active agent particles have a particlesize of less than about 5 microns.
 16. The method of claim 1, whereinthe level of redispersibility is sufficient for in vivo effectiveness ofthe dosage form if the active agent has an effective average particlesize prior to incorporation into the dosage form of less than about 800nm, and upon reconstitution in media representative of humanphysiological conditions the dosage form redisperses such that 90% ofthe active agent particles have a particle size of less than about 4microns.
 17. The method of claim 1, wherein the level ofredispersibility is sufficient for in vivo effectiveness of the dosageform if the active agent has an effective average particle size prior toincorporation into the dosage form of less than about 600 nm, and uponreconstitution in media representative of human physiological conditionsthe dosage form redisperses such that 90% of the active agent particleshave a particle size of less than about 3 microns.
 18. The method ofclaim 1, wherein the level of redispersibility is sufficient for in vivoeffectiveness of the dosage form if the active agent has an effectiveaverage particle size prior to incorporation into the dosage form ofless than about 400 nm, and upon reconstitution in media representativeof human physiological conditions the dosage form redisperses such that90% of the active agent particles have a particle size of less thanabout 2 microns.
 19. The method of claim 1, wherein the level ofredispersibility is sufficient for in vivo effectiveness of the dosageform if the active agent has an effective average particle size prior toincorporation into the dosage form of less than about 200 nm, and uponreconstitution in media representative of human physiological conditionsthe dosage form redisperses such that 90% of the active agent particleshave a particle size of less than about 1 micron.